A recent study showed that miR-26a is downregulated in hepatocellular carcinoma
tissues and that this downregulation is an independent predictor of survival.
Interestingly, the same study also reported that miR-26a downregulation causes a
concomitant elevation of IL-6 expression. Because miR-26a expression was found
to be transcriptionally downregulated by oncogene c-Myc in various cancers, and
the expression of c-Myc was increased by IL-6 stimulation, we hypothesized that
IL-6 contributes to reduction of miR-26a in hepatocellular carcinoma. Serum IL-6
was measured by ELISA and miR-26a was detected by qRT-PCR. The data of 30
patients with hepatocellular carcinoma who had undergone surgical tumor
resection revealed that serum IL-6 could be considered to be a predictor of
survival up to 5 years for hepatocellular carcinoma patients (log-rank test, P
< 0.05). We observed that the serum IL-6 concentration was inversely
correlated with miR-26a expression in cancerous tissues (Pearson correlation
test, r = -0.651, P < 0.01). Furthermore, by in vitro
experiments with HepG2 cells, we showed that IL-6 stimulation can lead to
miR-26a suppression via c-Myc activation, whereas in normal hepatocyte LO2 cells
incubation with IL-6 had no significant effect on miR-26a expression. Taken
together, these results indicate that miR-26a reduction in hepatocellular
carcinoma might be due to IL-6 upregulation.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate cellular gene
expression and are functionally linked to tumorigenesis (1). While some of the miRNAs are oncogenic, miRNAs such as
miR-26a are consistently downregulated in a number of cancers, demonstrating their
potential tumor suppressor functions (2,
3). Recently, a study by Ji et al. (4) revealed that miR-26a is downregulated in
hepatocellular carcinoma (HCC) and that this downregulation is associated with poor
survival. Therefore the authors indicated that the reduction of miR-26a expression
may be used as an independent predictor of survival for HCC patients. In the cited
study, the authors also found that the miR-26a reduction in cancerous tissues is
accompanied by a concomitant elevation in interleukin (IL)-6 expression (4). miR-26a expression has been found to be
transcriptionally downregulated by oncogene c-Myc in a variety of human cancers
3,
5,
6). Interestingly, several studies have
determined that IL-6 can induce a significant induction of c-Myc expression in
cancer cells including human HCC HepG2 cells (7-
9). Therefore, it is reasonable to
hypothesize that the miR-26a reduction in HCC is the consequence, at least in part,
of elevated IL-6 expression.

IL-6 is a multifunctional cytokine that was originally characterized as a regulator
of immune and inflammatory responses (10).
Recent evidence has revealed that IL-6 also plays a prominent role in the
pathogenesis of tumor cells (11,
12). Supporting the role of IL-6 in human
tumors is the observation of elevated serum concentrations of IL-6 and their
association with a poor clinical outcome (12,
13). In addition, IL-6 produced by tumor
cells also acts as a paracrine or autocrine regulatory factor and therefore
regulates tumor cell behaviors including metastasis, proliferation and survival
12,
14). The prognostic value of serum IL-6 has
been well demonstrated in various tumors. Furthermore some studies pointed out that
the prognostic value of serum IL-6 is independent of other known prognostic
variables (15-
17). The current study was designed to
examine the prognostic value of serum IL-6 regarding survival and the correlation
between serum IL-6 and tumor miR-26a expression in HCC patients. Furthermore, we
determined whether IL-6 stimulation can induce the reduction of miR-26a expression
via activation of c-Myc in HCC cells.

Material and Methods

Clinical samples

Thirty pairs of cancerous tissues and adjacent noncancerous tissues and matched
serum samples were collected from 30 patients with early stage HCC (21 cases of
stage I and 9 cases of stage II, determined according to criteria of the
International Union against Cancer) who underwent surgical tumor resection
between 2003 and 2005 at Xijing Hospital (Fourth Military Medical University,
Xi'an, China) and Southwest Hospital (Third Military Medical University,
Chongqing, China). None of the patients received neoadjuvant chemotherapy or
radiation therapy before surgery. The patients with confirmed cirrhosis or acute
infectious diseases (detectable serum hepatitis B virus (HBV) DNA or hepatitis C
virus RNA, and severe bacterial infection) were excluded from the study. All
patients studied were males, with a mean age of 46 years (range = 36-61 years).
Tissue samples were collected from each patient immediately after surgical
removal and snap-frozen in liquid nitrogen for subsequent analysis. Serum
samples were collected before surgery by low-speed centrifugation of heparinized
whole blood and stored in liquid nitrogen until assayed.

Follow-up assessments

Follow-up assessments were performed at annual intervals for up to 5 years after
surgery by phone and mail. The median follow-up time was 49.5 months (range = 9
to 60 months). Among the 30 patients who participated in this study, 11 patients
who survived longer than 5 years without recurrence after curative surgery for
HCC were classified as the no recurrence group, whereas the remaining 19
patients were classified as the recurrence group (survival <5 years for
recurrence and >5 years with recurrence).

Detection of serum IL-6

The concentration of serum IL-6 was determined in duplicate using commercially
available enzyme-linked immunosorbent assay kits (R&D Systems, Inc., USA)
according to manufacturer instructions.

Detection of miR-26a expression

Total RNA from frozen tissues or cells was isolated with Trizol (Takara, China)
according to manufacturer instructions. The TaqMan stem-loop RT-PCR method was
used to assess the expression of mature miR-26a (miR-26a-1) with kits from
Applied Biosystems (USA) as described previously (18). The real-time PCR results, recorded as threshold cycle numbers,
were normalized against an internal control (18S rRNA), and then reported as
fold-changes. Primers used for PCR amplification were: 5′-CCGCCGTTCAAGTAATCCAGGA-3′
and 5′-GTGCAGGGTCCGAGGT-3′.

Ethics statement

Ethics approval for the research was obtained from the Ethics Committee of Xijing
Hospital and Southwest Hospital, and written informed consent was obtained from
all patients who participated in the study.

Cell culture and IL-6 stimulation

Human HCC HepG2 cells and normal hepatocyte LO2 cells were routinely cultured in
RPMI 1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum
(HyClone) at 37°C in a 5% CO2 incubator. For stimulation, cells
were starved with serum-free medium for 6 h and then incubated with different
doses of human recombinant IL-6 (Sigma-Aldrich, USA) for 2 h, after which total
proteins and RNA were extracted. For the blocking assay, HepG2 cells were
pre-incubated with 100 µM of c-Myc inhibitor 10058-F4 (Sigma-Aldrich) for
6 h before IL-6 stimulation (19).

Cell transfection

LO2 cells transfected with plasmids expressing c-Myc (pcDNA3.1-c-Myc, provided by
Dr. Xiaohua Li, State Key Laboratory of Cancer Biology and Xijing Hospital of
Digestive Diseases, Fourth Military Medical University, Xi'an, China) were
selected with medium containing G418 (Sigma-Aldrich) for 6 weeks to generate a
stable cell line with higher expression of c-Myc. LO2 cells transfected with
blank pcDNA3.1 plasmids were used as control.

Western blot analysis

Total cell lysates were prepared in 1X SDS buffer. Proteins were separated by
SDS-PAGE and transferred to PVDF membranes. Membranes were then blotted with
antibodies specific for c-Myc and β-actin (Sigma-Aldrich). Antigen-antibody
complexes were visualized using enhanced chemiluminescence (Sigma-Aldrich).

Statistical analysis

Data are reported as means ± SEM. Differences were compared by one-way ANOVA
followed by the least significant difference t-test.
Correlations between two groups were assessed by the Pearson correlation test.
Survival curves were plotted by the Kaplan-Meier method and the log-rank test
was carried out to compare differences in survival. All statistical analyses
were performed using the Prism 5.0 software (GraphPad Software Inc., USA). P
< 0.05 was considered to be statistically significant.

Results

Elevated serum IL-6 is a useful predictor of recurrence after curative
resection of HCC

In this study, we compared the serum IL-6 concentration between two patient
groups with or without recurrence after surgery of early stage HCC. As shown in
Figure 1A, the concentrations of serum
IL-6 determined prior to surgery in patients who developed recurrence were
significantly higher than those of patients without recurrence during 5 years of
follow-up (21.45 ± 6.98 vs 16.09 ± 5.87, P <
0.05). We further assessed the correlation between serum IL-6 and survival time.
As shown in Figure 1B, patients with a
high serum IL-6 concentration had significantly shorter survival times than
those with a low serum IL-6 concentration (log-rank test, P < 0.05). These
results indicate that a high serum IL-6 concentration is associated with
postoperative recurrence and can be used as a predictor for survival of patients
with HCC.

Figure 1 Serum IL-6 is a predictor of survival in hepatocellular carcinoma
patients. A, Comparison of serum IL-6 concentration
with recurrence (least significant difference t-test).
The number of patients is given in parentheses. Recurrence of
hepatocellular carcinoma was based on image observations and the
clinical course. B, Survival curve of patients who
underwent curative resection of hepatocellular carcinoma. A median
expression level was used as the cutoff. Kaplan-Meier analysis and the
log-rank test showed that the survival rate of the group with serum IL-6
concentrations lower than 18 pg/mL was more favorable than that of the
group with serum IL-6 higher than 18 pg/mL (P < 0.05).

In this study, we hypothesized that elevated IL-6 expression contributes to the
reduction of miR-26a expression in HCC through activation of the c-Myc pathway.
We tested the effect of IL-6 stimulation on miR-26a expression in cultured HCC
HepG2 cells. As previously reported, c-Myc protein was increased in a
dose-dependent manner after 2-h incubation with IL-6 (Figure 3A). More importantly, the expression of miR-26a in
HepG2 cells was decreased in response to incubation with IL-6 (Figure 3B). In order to further elucidate
whether c-Myc was involved in the decreased expression of miR-26a upon IL-6
stimulation, a small molecule c-Myc inhibitor, 10058-F4, was used to examine the
effect of c-Myc inhibition on IL-6-induced downregulation of miR-26a. As
expected, the c-Myc inhibitor blocked IL-6/miR-26a signaling (Figure 3B). Taken together, these results
support the view that IL-6 suppresses the expression of miR-26a via c-Myc
activation in HCC cells.

Figure 3 IL-6 stimulation leads to the reduction of miR-26a expression via
activation of c-Myc in HepG2 cells. A, Representative
immunoblots showed that c-Myc protein in HepG2 cells was increased in a
dose-dependent manner upon IL-6 stimulation. β-actin was used as an
internal control. B, Quantitative RT-PCR showed that
miR-26a expression was reduced upon IL-6 stimulation, and the reduced
miR-26a expression could be blocked by a c-Myc inhibitor (10058-F4). The
data are representative of five independent experiments.

IL-6 incubation has no significant effect on the expression of miR-26a in
normal hepatocyte LO2 cells

The Pearson correlation test showed that there was no significant correlation
between serum IL-6 concentration and miR-26a expression in adjacent noncancerous
hepatic tissues. To verify this, we further analyzed the effect of IL-6
stimulation on the expression of miR-26a in normal human hepatocyte LO2 cells.
As shown in Figure 4A, the expression of
miR-26a in LO2 cells did not change significantly after incubation with IL-6. We
also determined the protein concentration of c-Myc in LO2 cells after IL-6
incubation. The results of Western blot analysis showed that there were also no
significant changes in c-Myc expression (Figure
4B), indicating that in normal hepatocyte cells IL-6 stimulation
cannot activate c-Myc and thus suppress the expression of miR-26a. To determine
whether or not c-Myc activation can lead to miR-26a reduction in normal
hepatocyte cells, LO2 cells with increased c-Myc protein were selected
(LO2-pc-c-Myc cells, Figure 4C) and
analyzed for miR-26a expression. Also, as shown in Figure 4D, no obvious miR-26 reduction was found in LO2
cells with upregulated c-Myc protein expression. These results suggest that the
regulation of miR-26a expression in normal hepatocyte cells might be influenced
by factors that differ from those affecting HCC cells.

Figure 4 IL-6 stimulation does not affect the expression of miR-26a in LO2
cells. A, Quantitative RT-PCR showed that miR-26a
expression in LO2 cells was not modified upon IL-6 stimulation (N = 5, P
> 0.05). B, Expression of c-Myc protein in
LO2-pc-c-Myc cells. C, Representative immunoblots also
showed that c-Myc expression was not changed by IL-6.
D, Expression of miR-26a in LO2-pc-c-Myc cells.
Compared with the expression of miR-26a in control LO2-pcDNA3.1 cells,
the expression of miR-26a in LO2-pc-c-Myc cells was not significantly
modified (N = 5, P > 0.05).

Discussion

IL-6 has many physiologic roles and has been implicated in the process of
tumorigenesis. A great variety of tumor types are stimulated by IL-6, including
melanoma, Kaposi's sarcoma, lymphoma and leukemia, multiple myeloma, renal cell
carcinoma, ovarian carcinoma, prostate carcinoma, colorectal carcinoma, and HCC
20). The ability of IL-6 to promote
transcription of oncogene c-Myc in HCC HepG2 cells has been reported (9). Moreover, Chang et al. (3) recently demonstrated that hyperactivity of c-Myc leads to
repression of miR-26a expression in various human cancer cells. Therefore, it is
possible that elevated IL-6 expression increases the activity of c-Myc via paracrine
or autocrine mechanisms and consequently leads to the reduction of miR-26a
expression.

Several studies have revealed that serum IL-6 concentration is elevated in patients
with HCC and that it can be used as a tumor marker, even more efficient than
alpha-fetoprotein (AFP) (21-
24). There are controversial reports about
the significance of the use of serum IL-6 concentration for predicting survival.
Giannitrapani et al. (21) reported that serum
IL-6 has a significant positive correlation with the stage of HCC. Malaguarnera et
al. (22) found that there is an elevated
positive correlation between serum IL-6 and the size of HCC. Because the stage and
size of tumors are both factors significantly associated with survival (25), it would be reasonable to propose that
serum IL-6 concentration is also associated with survival in patients with HCC.
However, Chau et al. (23) reported that there
is no significant correlation between serum IL-6 and survival in HCC patients. It is
interesting to note that the patients involved in the study of Chau et al. (23) were all from Chinese Taipei. Because most
of the patients with HCC in China are HBV-related, and a large proportion of them
have developed liver cirrhosis at the time of diagnosis, it might be difficult to
evaluate the significance of serum IL-6 in predicting survival if cirrhosis-related
death is not excluded (26-
28). Gender is an important deciding factor
for the expression of IL-6. Many studies have shown that estrogen inhibits IL-6
expression, which now is thought to be a key reason for the decreased susceptibility
to HCC in females (29). Moreover, serum IL-6
concentration is affected by various clinical conditions, especially acute
inflammation, which usually causes a significant increase in IL-6 expression (30). Due to the confounding factors discussed
above, we did not find the predicted value of serum IL-6 in all of the 51 cases,
which were initially included in this study (data not shown). However, after
excluding female patients, patients with cirrhosis and acute infection from the
statistical analysis, we observed that elevated serum IL-6 concentration can be
considered to be a predictive risk factor for recurrence and reduced survival in our
patients with HCC.

The oncogene c-Myc is pathologically activated in many human malignancies. Recent
studies have revealed that activation of c-Myc may induce a widespread repression of
miRNA expression (3). MiR-26a was shown to be
repressed by c-Myc in almost all c-Myc-dependent tumors examined, thereby
representing one of the most interesting c-Myc-repressed miRNAs (3,
5,
6). Sander et al. (5) and Kota et al. (31
reported that the reduction of miR-26a expression plays an important role in
mediating c-Myc-dependent cell proliferation. The study of Kota et al. (31) in HepG2 cells also suggested that the
reduction of miR-26a expression is involved in the apoptosis-resistant phenotype
mediated by c-Myc. These observations indicated that miR-26a may be deeply involved
in the transduction of the oncogenic c-Myc signal. Nevertheless, in our study, we
also observed that in normal hepatocyte LO2 cells IL-6 incubation cannot stimulate
c-Myc activation and lead to miR-26a suppression. This is probably due to the fact
that c-Myc expression is controlled by a large number of signaling pathways in
normal cells (32), which therefore protect
normal cells from c-Myc-mediated oncogenesis.

We demonstrated that IL-6 upregulation contributes to the reduction of miR-26a
expression in HCC tissues. Because serum IL-6 concentration can be used as a
valuable prognostic indicator for patients with HCC, it may not be advisable to view
miR-26a as an independent predictor of survival, and reduction of miR-26a expression
should also be viewed as a poor prognostic marker.

Acknowledgements

Research supported by the National Foundation of Natural Sciences of China (#81071727
and #81101533), China Postdoctoral Science Foundation (#20100481468 and #201104755)
and Natural Science Foundation of Hubei Province of China (#2011CDB017).